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Abstract In vitro maturation of mammalian oocytes is a key step during in vitro production of mammalian embryos. Studies have shown that the level of oocyte maturation is positively correlated with embryo cleavage rate and blastocyst rate. However， reactive oxygen species （ROS） in vitro is one of the main influencing factors of oocyte maturation. The results indicate that the addition of antioxidants can effectively improve in vitro oocyte maturation level and reduce the adverse effects of reactive oxygen species， which contribute to oocyte development. This paper summarizes research progresses on the effects of different antioxidants on in vitro development of mammalian oocytes， which is conducive to further analysis of the mechanism of action.
Key words Mammalian oocyte； In vitro development； Antioxidants
Oxidative stress induced by the accumulation of reactive oxygen species is the main reason for oocyte death and damage during in vitro culture of mammalian oocytes[1-2]. In the process of in vitro oocyte maturation， changes in light， oxygen concentration， metabolism rate and enzyme content may enhance ROS levels. Excessive ROS may destroy gametocyte membranes and affect metabolism and gene expression patterns. ROS-induced oxidative stress causes mitochondrial dysfunction， while inhibiting sperm and egg fusion[3-5]. Reactive oxygen species have been identified as the main factors that reduce the rate of oocyte development in vitro[6]. Adding antioxidants in in vitro culture medium can reduce ROS levels and improve cell development[7-8].
Overview of Roles of Antioxidants
Antioxidants are a type of compounds that react with free radicals to reduce the free radicals into non-free radicals. Both exogenous and endogenous antioxidants can eliminate free radicals through redox reactions. In recent years， with continuous improvement of in vitro oocyte culture system， antioxidants are gradually being applied to actual production. In general， antioxidants can be divided into two categories： enzymatic antioxidants and non-enzymatic antioxidants. Enzymatic antioxidants include superoxide dismutase （SOD）， catalase （CAT） and E-64， which can resist oxidative stress by increasing the enzyme activity. Non-enzymatic antioxidants include proanthocyanidins， melatonin， inositol and Ge-132.
Mechanisms of Action of Non-enzymatic Antioxidants
Proanthocyanidins
Proanthocyanidins are a type of natural polyphenolics widely found in plants such as grape seeds and hawthorn， which have extremely strong antioxidant capacity and free radical scavenging ability[9]. Proanthocyanidins are polymers formed by the condensation of different amounts of catechin or epicatechin， and their dimers have strong antioxidant activity. Nrf2 is a key factor in cellular oxidative stress response， which can regulate encoding protective proteins by interacting with the antioxidant element ARE[10]. Protective proteins include antioxidant proteins and phase II detoxification enzymes， such as heme oxygenase-1 （HO-1）， quinone oxidoreductase 1 （NQO1）， and glutathione S-transferase （GST）[11]. According to foreign literature[12-13]， treating oxidatively damaged animals with proanthocyanidin can enhance the expression levels of Nrf2 and downstream HO-1 and NQO1 in vitro. Feng et al.[14] found that proanthocyanidin could resist oxidative damage by activating Nrf2 signaling pathway. Adding appropriate levels of proanthocyanidins during in vitro culture of oocytes can increase in vitro developmental capacity of oocytes and protect them from oxidative damage[14].
L-ergothioneine （LE）
L-ergothioneine is a rare natural amino acid， which acts as a potent OH-scavenger and an inhibitor of iron and copper ions， thus inhibiting H2O2 generation. LE is synthesized by some bacteria and fungi rather than animals. Therefore， mammals can only obtain LE from their diet. Moreover， LE clears singlet oxygen， hydroxyl radicals， hypochlorous acid and reactive oxygen species， and inhibits peroxynitrite nitration of proteins and DNA. Studies have shown that LE has higher antioxidant activity that can remove free radicals more effectively compared with traditional antioxidants. It has been reported that the addition of LE during in vitro maturation （IVM）， in vitro fertilization （IVF） and in vitro culture （IVC） can significantly improve in vitro maturation rate of ovine oocytes[15].
Inositol
Inositol， similar to glucose in structure， is an growth factor of animals and plants. The addition of MI in the culture liquid can improve in vitro blastocyst rate in cattle[16]. Adding inositol in in vitro culture medium can improve oocyte maturation rate in mice. In previous studies， IVF operation was conducted with oocytes obtained from female ovaries. Oocytes with good quality were used in group A； oocytes with poor quality were used in group B. The results show that MI level in follicular fluid of group A is significantly higher than that of group B， but there is no significant difference in MI level in blood between two groups. Oocytes with better morphology contain more MI in follicular fluid， and there is a positive correlation between MI levels and fertilization rate. In previous animal models， MI concentration in follicular fluid could be increased by active transport[17]. These results indicate that MI can improve oocyte maturation rate. Melatonin
Melatonin is a type of neuroendocrine hormone secreted by the pineal gland， which exerts strong anti-oxidative effects such as directly scavenging free radicals， improving antioxidant enzyme activity and reducing free radical generation， while having fewer side effects than other hormones with many advantages compared with traditional antioxidants.
During in vitro maturation of porcine oocytes， exogenous melatonin can promote nuclear and cytoplasmic maturation of oocytes. The addition of 10-9 mol/L melatonin to the medium could significantly improve porcine oocyte maturation rate and increase the number of parthenogenetic blastocysts[18]. After in vitro culture， treating porcine oocytes under heat stress with 10-7 mol/L Mel could enhance intracellular GSH levels. Takada et al.[19] confirmed that higher concentrations of melatonin could improve the efficiency of oocyte maturation during meiosis metaphase. Takada et al.[20] found that H2O2 might prevent oocyte maturation in mice， while melatonin could effectively reduce the level of hydrogen peroxide in oocytes. Melatonin can effectively reduce intracellular DNA damage caused by oxidative stress， inhibit apoptosis and promote normal development of oocytes.
Ge-132
Germanium （Ge） is one of trace elements. Organogermanium compounds， such as carboxyethylgermanium sesquioxide （Ge-132）， exhibit low toxicity and increasing biological activity. The experimental results show that in vitro culture of porcine oocytes by Ge-132 treatment can significantly enhance the developmental potential of in vitro culture of porcine oocytes and increase intracellular glutathione levels， so as to decrease intracellular ROS levels and reduce oxidative stress-induced apoptosis， thereby regulating mRNA expression level during cell maturation.
Mechanisms of Action of Enzymatic Antioxidants
There are a variety of antioxidant enzymes in mammalian oocytes， which are activated during different developmental stages of oocytes， thus exerting specific anti-oxidative activities.
E-64
The maturation and development of oocytes are closely related to cumulus cells. During the process of oocyte maturation， cumulus cells play an important role in nutrition and protection of oocytes. There is a microvilli structure between cumulus cells and oocytes. Amino acids and other small molecules can be transported directly into oocyte cytoplasm， thus promoting oocyte maturation. Therefore， the proliferation of cumulus cells is generally regarded as one of the important factors that reflect the level of in vitro maturation and development of oocytes. Ballboula et al.[21] reported that adding E-64 to mature liquid could significantly reduce the apoptosis rate of cumulus cells， oocytes and embryos. These studies indicate that protease E-64 may play a role in improving in vitro development ability of oocytes by reducing cumulus cell apoptosis or directly acting on oocytes. Lonergan et al. found that embryonic development ability is an important indicator reflecting the quality of oocytes. It has been confirmed that adding different concentrations of E-64 in mature culture medium can significantly enhance in vitro maturation rate of porcine oocytes， in vitro developmental ability of embryos and developmental rate of parthenogenetic blastocysts. Studies have shown that E-64 can significantly enhance blastocyst rate of porcine oocytes while exerting no significant effect on cleavage rate of porcine oocytes， which indicates that E-64 can effectively promote the subsequent developmental ability of porcine oocytes. Si et al. found that adding 10μmol/L E-64 in in vitro culture medium could result in remarkably higher maturity and nuclear maturity compared with the non-addtion group and significantly improve porcine parthenogenic oocyte cleavage rate and blastocyst rate. The optimal concentration of E-64 in mature liquid was 10μmol/L， which could significantly improve cleavage rate， nuclear maturation rate and blastocyst rate of porcine oocytes.
Superoxide dismutase （SOD）
Superoxide dismutase （SOD） can be divided into three types according to different metal cofactors. Type 1： Cu.Zn-SOD， most common， green， exists mainly in the cytoplasm； type 2： Mn-SOD， purple， exists in eukaryotic mitochondria and prokaryotic cells； type 3： Fe-SOD， brown， present in prokaryotic cells.
SOD is an important antioxidant enzyme in mammalian oocytes. The level of SOD in vivo is an intuitive indicator of aging and death. So far， it has been confirmed that more than 60 kinds of diseases are caused by oxygen free radicals. SOD can antagonize and block the damage caused by oxygen free radicals to oocytes， repair damaged oocytes in time， and restore free radical-induced damage of oocytes[22].
Agricultural Biotechnology2018
Conclusion
After continuous research and exploration， it has been confirmed that the developmental ability of mammalian oocytes can be significantly improved by adding different types of antioxidants during in vitro culture of mammalian oocytes. Thus， the existing maturation system of mammalian oocytes in vitro has been further optimized. Although it has not reached the closest level to culture environment in vivo， it is believed that with the continuous improvement of technologies， a large number of high-quality mammalian oocytes will be obtained for actual production. References
[1] Karuputhula NB， Chattopadhyay R， Chakravarty B， et al. Oxidative status in granulosa cells of infertile women undergoing IVF[J]. Systems Biology in Reproductive Medicine， 2013， 59（2）：91-98.
[2] Tamura H， Takasaki A， Miwa I， et al. Oxidative stress impairs oocyte quality and melatonin protects oocytes from free radical damage and improves fertilization rate[J]. J. Pineal Res.， 2008， 44：280-287.
[3] Aitken RJ， Clarkson JS. Cellular basis of defective sperm function and its association with the genesis of reactive oxygen species by human spermatozoa[J]. J. Reprod. Fertil.， 1987， 81：459-469.
[4] Goncalves FS， Barretto LS， Arruda RP， et al. Effect of antioxidants during bovine in vitro fertilization procedures on spermatozoa and embryo development[J]. Reprod. Domest. Anim.， 2010， 45：129-135.
[5] Takahashi M. Oxidative stress and redox regulation on in vitro development of mammalian embryos[J]. J. Reprod. Dev.， 2012， 58：1-9.
[6] Wang F， Tian X， Zhang L， et al. Beneficial effects of melatonin on in vitro bovine embryonic development are mediated by melatonin receptor 1[J]. J. Pineal Res.， 2014， 56：333-342.
[7] Nabenishi H， Ohta H， Nishimoto T， et al. The effects of cysteine addition during in vitro maturation on the developmental competence， ROS GSH and apoptosis level of bovine oocytes exposed to heat stress[J]. Zygote， 2012， 20：249-259.
[8] Wang F， Tian X， Zhang L， et al. Beneficial effect of resveratrol on bovine oocyte maturation and subsequent embryonic development after in vitro fertilization[J]. Fertil. Steril.， 2014， 101：577-586.
[9] Taheri R， Connolly BA， Brand MH， et al. Underutilize chokeberry accessions are rich sources of anthocyanidins， flavonoids， hydroxycinnamic acids and proanthocyanidins[J]. J Agric Food Chem， 2013， 26： 2569-2574.
[10] Kwak MK， Wakabayashi N， Kensler TW. Chemoprevention through the Keap1-Nrf2 signaling pathway by phase 2 enzyme inducers[J]. Mutation Res， 2004， 555：133-148.
[11] Kim SK， Yang JW， Kim MR， et al. Increased expression of Nrf2-ARE-dependent anti-oxidant in tamoxifen-resistant breast cancer cells[J]. Free Radic Biol Med， 2008， 45： 537-546.
[12] CHEN Y， ZHANG SL， JIANG J， et al. Protective effect and mechanism of grape seed procyanidins extract on oxidative injury of kidney induced by food-borne lead in rat[J]. China Feed， 2016， 4：19-22.（in Chinese）
[13] Sun Y， Xiu CM， Liu W， et al. Grape seed proanthocyanidin extract protects the retina against early diabetic injury by activating the Nrf2 pathway[J]. ExpTher Med， 2016， 11：1253-1258.
Key words Mammalian oocyte； In vitro development； Antioxidants
Oxidative stress induced by the accumulation of reactive oxygen species is the main reason for oocyte death and damage during in vitro culture of mammalian oocytes[1-2]. In the process of in vitro oocyte maturation， changes in light， oxygen concentration， metabolism rate and enzyme content may enhance ROS levels. Excessive ROS may destroy gametocyte membranes and affect metabolism and gene expression patterns. ROS-induced oxidative stress causes mitochondrial dysfunction， while inhibiting sperm and egg fusion[3-5]. Reactive oxygen species have been identified as the main factors that reduce the rate of oocyte development in vitro[6]. Adding antioxidants in in vitro culture medium can reduce ROS levels and improve cell development[7-8].
Overview of Roles of Antioxidants
Antioxidants are a type of compounds that react with free radicals to reduce the free radicals into non-free radicals. Both exogenous and endogenous antioxidants can eliminate free radicals through redox reactions. In recent years， with continuous improvement of in vitro oocyte culture system， antioxidants are gradually being applied to actual production. In general， antioxidants can be divided into two categories： enzymatic antioxidants and non-enzymatic antioxidants. Enzymatic antioxidants include superoxide dismutase （SOD）， catalase （CAT） and E-64， which can resist oxidative stress by increasing the enzyme activity. Non-enzymatic antioxidants include proanthocyanidins， melatonin， inositol and Ge-132.
Mechanisms of Action of Non-enzymatic Antioxidants
Proanthocyanidins
Proanthocyanidins are a type of natural polyphenolics widely found in plants such as grape seeds and hawthorn， which have extremely strong antioxidant capacity and free radical scavenging ability[9]. Proanthocyanidins are polymers formed by the condensation of different amounts of catechin or epicatechin， and their dimers have strong antioxidant activity. Nrf2 is a key factor in cellular oxidative stress response， which can regulate encoding protective proteins by interacting with the antioxidant element ARE[10]. Protective proteins include antioxidant proteins and phase II detoxification enzymes， such as heme oxygenase-1 （HO-1）， quinone oxidoreductase 1 （NQO1）， and glutathione S-transferase （GST）[11]. According to foreign literature[12-13]， treating oxidatively damaged animals with proanthocyanidin can enhance the expression levels of Nrf2 and downstream HO-1 and NQO1 in vitro. Feng et al.[14] found that proanthocyanidin could resist oxidative damage by activating Nrf2 signaling pathway. Adding appropriate levels of proanthocyanidins during in vitro culture of oocytes can increase in vitro developmental capacity of oocytes and protect them from oxidative damage[14].
L-ergothioneine （LE）
L-ergothioneine is a rare natural amino acid， which acts as a potent OH-scavenger and an inhibitor of iron and copper ions， thus inhibiting H2O2 generation. LE is synthesized by some bacteria and fungi rather than animals. Therefore， mammals can only obtain LE from their diet. Moreover， LE clears singlet oxygen， hydroxyl radicals， hypochlorous acid and reactive oxygen species， and inhibits peroxynitrite nitration of proteins and DNA. Studies have shown that LE has higher antioxidant activity that can remove free radicals more effectively compared with traditional antioxidants. It has been reported that the addition of LE during in vitro maturation （IVM）， in vitro fertilization （IVF） and in vitro culture （IVC） can significantly improve in vitro maturation rate of ovine oocytes[15].
Inositol
Inositol， similar to glucose in structure， is an growth factor of animals and plants. The addition of MI in the culture liquid can improve in vitro blastocyst rate in cattle[16]. Adding inositol in in vitro culture medium can improve oocyte maturation rate in mice. In previous studies， IVF operation was conducted with oocytes obtained from female ovaries. Oocytes with good quality were used in group A； oocytes with poor quality were used in group B. The results show that MI level in follicular fluid of group A is significantly higher than that of group B， but there is no significant difference in MI level in blood between two groups. Oocytes with better morphology contain more MI in follicular fluid， and there is a positive correlation between MI levels and fertilization rate. In previous animal models， MI concentration in follicular fluid could be increased by active transport[17]. These results indicate that MI can improve oocyte maturation rate. Melatonin
Melatonin is a type of neuroendocrine hormone secreted by the pineal gland， which exerts strong anti-oxidative effects such as directly scavenging free radicals， improving antioxidant enzyme activity and reducing free radical generation， while having fewer side effects than other hormones with many advantages compared with traditional antioxidants.
During in vitro maturation of porcine oocytes， exogenous melatonin can promote nuclear and cytoplasmic maturation of oocytes. The addition of 10-9 mol/L melatonin to the medium could significantly improve porcine oocyte maturation rate and increase the number of parthenogenetic blastocysts[18]. After in vitro culture， treating porcine oocytes under heat stress with 10-7 mol/L Mel could enhance intracellular GSH levels. Takada et al.[19] confirmed that higher concentrations of melatonin could improve the efficiency of oocyte maturation during meiosis metaphase. Takada et al.[20] found that H2O2 might prevent oocyte maturation in mice， while melatonin could effectively reduce the level of hydrogen peroxide in oocytes. Melatonin can effectively reduce intracellular DNA damage caused by oxidative stress， inhibit apoptosis and promote normal development of oocytes.
Ge-132
Germanium （Ge） is one of trace elements. Organogermanium compounds， such as carboxyethylgermanium sesquioxide （Ge-132）， exhibit low toxicity and increasing biological activity. The experimental results show that in vitro culture of porcine oocytes by Ge-132 treatment can significantly enhance the developmental potential of in vitro culture of porcine oocytes and increase intracellular glutathione levels， so as to decrease intracellular ROS levels and reduce oxidative stress-induced apoptosis， thereby regulating mRNA expression level during cell maturation.
Mechanisms of Action of Enzymatic Antioxidants
There are a variety of antioxidant enzymes in mammalian oocytes， which are activated during different developmental stages of oocytes， thus exerting specific anti-oxidative activities.
E-64
The maturation and development of oocytes are closely related to cumulus cells. During the process of oocyte maturation， cumulus cells play an important role in nutrition and protection of oocytes. There is a microvilli structure between cumulus cells and oocytes. Amino acids and other small molecules can be transported directly into oocyte cytoplasm， thus promoting oocyte maturation. Therefore， the proliferation of cumulus cells is generally regarded as one of the important factors that reflect the level of in vitro maturation and development of oocytes. Ballboula et al.[21] reported that adding E-64 to mature liquid could significantly reduce the apoptosis rate of cumulus cells， oocytes and embryos. These studies indicate that protease E-64 may play a role in improving in vitro development ability of oocytes by reducing cumulus cell apoptosis or directly acting on oocytes. Lonergan et al. found that embryonic development ability is an important indicator reflecting the quality of oocytes. It has been confirmed that adding different concentrations of E-64 in mature culture medium can significantly enhance in vitro maturation rate of porcine oocytes， in vitro developmental ability of embryos and developmental rate of parthenogenetic blastocysts. Studies have shown that E-64 can significantly enhance blastocyst rate of porcine oocytes while exerting no significant effect on cleavage rate of porcine oocytes， which indicates that E-64 can effectively promote the subsequent developmental ability of porcine oocytes. Si et al. found that adding 10μmol/L E-64 in in vitro culture medium could result in remarkably higher maturity and nuclear maturity compared with the non-addtion group and significantly improve porcine parthenogenic oocyte cleavage rate and blastocyst rate. The optimal concentration of E-64 in mature liquid was 10μmol/L， which could significantly improve cleavage rate， nuclear maturation rate and blastocyst rate of porcine oocytes.
Superoxide dismutase （SOD）
Superoxide dismutase （SOD） can be divided into three types according to different metal cofactors. Type 1： Cu.Zn-SOD， most common， green， exists mainly in the cytoplasm； type 2： Mn-SOD， purple， exists in eukaryotic mitochondria and prokaryotic cells； type 3： Fe-SOD， brown， present in prokaryotic cells.
SOD is an important antioxidant enzyme in mammalian oocytes. The level of SOD in vivo is an intuitive indicator of aging and death. So far， it has been confirmed that more than 60 kinds of diseases are caused by oxygen free radicals. SOD can antagonize and block the damage caused by oxygen free radicals to oocytes， repair damaged oocytes in time， and restore free radical-induced damage of oocytes[22].
Agricultural Biotechnology2018
Conclusion
After continuous research and exploration， it has been confirmed that the developmental ability of mammalian oocytes can be significantly improved by adding different types of antioxidants during in vitro culture of mammalian oocytes. Thus， the existing maturation system of mammalian oocytes in vitro has been further optimized. Although it has not reached the closest level to culture environment in vivo， it is believed that with the continuous improvement of technologies， a large number of high-quality mammalian oocytes will be obtained for actual production. References
[1] Karuputhula NB， Chattopadhyay R， Chakravarty B， et al. Oxidative status in granulosa cells of infertile women undergoing IVF[J]. Systems Biology in Reproductive Medicine， 2013， 59（2）：91-98.
[2] Tamura H， Takasaki A， Miwa I， et al. Oxidative stress impairs oocyte quality and melatonin protects oocytes from free radical damage and improves fertilization rate[J]. J. Pineal Res.， 2008， 44：280-287.
[3] Aitken RJ， Clarkson JS. Cellular basis of defective sperm function and its association with the genesis of reactive oxygen species by human spermatozoa[J]. J. Reprod. Fertil.， 1987， 81：459-469.
[4] Goncalves FS， Barretto LS， Arruda RP， et al. Effect of antioxidants during bovine in vitro fertilization procedures on spermatozoa and embryo development[J]. Reprod. Domest. Anim.， 2010， 45：129-135.
[5] Takahashi M. Oxidative stress and redox regulation on in vitro development of mammalian embryos[J]. J. Reprod. Dev.， 2012， 58：1-9.
[6] Wang F， Tian X， Zhang L， et al. Beneficial effects of melatonin on in vitro bovine embryonic development are mediated by melatonin receptor 1[J]. J. Pineal Res.， 2014， 56：333-342.
[7] Nabenishi H， Ohta H， Nishimoto T， et al. The effects of cysteine addition during in vitro maturation on the developmental competence， ROS GSH and apoptosis level of bovine oocytes exposed to heat stress[J]. Zygote， 2012， 20：249-259.
[8] Wang F， Tian X， Zhang L， et al. Beneficial effect of resveratrol on bovine oocyte maturation and subsequent embryonic development after in vitro fertilization[J]. Fertil. Steril.， 2014， 101：577-586.
[9] Taheri R， Connolly BA， Brand MH， et al. Underutilize chokeberry accessions are rich sources of anthocyanidins， flavonoids， hydroxycinnamic acids and proanthocyanidins[J]. J Agric Food Chem， 2013， 26： 2569-2574.
[10] Kwak MK， Wakabayashi N， Kensler TW. Chemoprevention through the Keap1-Nrf2 signaling pathway by phase 2 enzyme inducers[J]. Mutation Res， 2004， 555：133-148.
[11] Kim SK， Yang JW， Kim MR， et al. Increased expression of Nrf2-ARE-dependent anti-oxidant in tamoxifen-resistant breast cancer cells[J]. Free Radic Biol Med， 2008， 45： 537-546.
[12] CHEN Y， ZHANG SL， JIANG J， et al. Protective effect and mechanism of grape seed procyanidins extract on oxidative injury of kidney induced by food-borne lead in rat[J]. China Feed， 2016， 4：19-22.（in Chinese）
[13] Sun Y， Xiu CM， Liu W， et al. Grape seed proanthocyanidin extract protects the retina against early diabetic injury by activating the Nrf2 pathway[J]. ExpTher Med， 2016， 11：1253-1258.